**4. Conclusions**

Here, we demonstrate that the minimum number of origins (MO) required to duplicate an entire chromosome within the S-phase duration can be easily obtained from the parameters chromosome size, S-phase duration, and replication rate. Predictions performed by us suggest that in the presence of replication stress, all the organisms analyzed here demands higher MO values. Moreover, we evidenced here that the MO allows the establishment of a threshold that can serve as a parameter by other methods that detect origins. Also, our data strongly suggest that trypanosomatids can use around fivefold more origins than the MO. This value is relatively higher than other single-celled organisms, such as the yeasts *S. cerevisiae* and *S. pombe*. However, further studies are required to figure out the dynamics of origin usage during the S-phase in these organisms, especially in trypanosomatids.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/5/523/s1, Figure S1: Parameters required to use the CeCyD website.

**Author Contributions:** M.S.d.S. conceived the rationale of the experimental design and this manuscript, with fundamental insights from M.C.E. M.S.d.S., and M.O.V. carried out the experiments. B.F.S. and M.O.V. developed the CeCyD website with insights from M.S.d.S. M.S.d.S. wrote the manuscript with essential contribution from M.O.V., B.F.S., and M.C.E. All authors read and approved the final version of the manuscript. M.C.E. supervised the project.

**Funding:** This research was funded by São Paulo Research Foundation (FAPESP) and Center of Toxins, Immune Response, and Cell Signaling (CeTICS) under grants 2013/07467-1, 2016/50050-2, 2014/24170-5, 2017/18719-2, 2017/07693-2). MCE is also fellow from the National Council for Scientific and Technological Development (CNPq) under grant 306199/2018-1 and MOV was fellow from CNPq under grant 870219/1997-9.

**Acknowledgments:** The authors thank the São Paulo Research Foundation (FAPESP) and Center of Toxins, Immune Response, and Cell Signaling (CeTICS) for their support.

**Conflicts of Interest:** The authors declare no conflict of interest.
